Adjuvant Properties of Lipopolysaccharide from Neisseria Meningitidis Serogroup B Detoxiﬁed and Conjugated with Tetanus Toxoid Julio A

Vaccine 24S2 (2006) S2/63–S2/64

Adjuvant properties of lipopolysaccharide from Neisseria meningitidis serogroup B detoxiﬁed and conjugated with tetanus toxoid Julio A. Balboa a, ∗, Maribel Cuello a, Osmir Cabrera a, Judith del Campo a, Miriam Lastre a, Danay Gil a, Carlos Taboada a, Mildrey Farinas˜ b, Marixa Hernandez´ c, Oliver Perez´ a

a Immunology Department, Finlay Institute, P.O. Box 16017, Havana, Cuba b Preclinic and Toxicological Department, Finlay Institute, P.O. Box 16017, Havana, Cuba c Quality Direction, Finlay Institute, P.O. Box 16017, Havana, Cuba

Available online 17 February 2005

Abstract

We evaluated the adjuvant properties and toxicity of purified Neisseria meningitidis serogroup B lipopolysaccharide (LPS) conjugated with tetanus toxoid (TT) using a new method of conjugation to obtain amine groups in the polysaccharide structure. The endotoxic activity of treated LPS was reduced 2400 times as determined by Limulus amoebocyte assay and no mortality was observed in Balb/c mice inoculated with detoxified LPS versus 100% mortality in native LPS inoculated mice. The conjugated LPS–TT elicited in mice higher anti-TT IgG2a and IgG1 than unconjugated TT. In addition, high levels of anti-LPS IgG and IgG subclasses were detected in sera. These results evidence the adjuvant activity of detoxified LPS and may suggest that the conjugation to TT changes the LPS immune response from thymus-independent to thymus-dependent. © 2005 Elsevier Ltd. All rights reserved.

Keywords: Lipopolysaccharide; Conjugate; Adjuvant; Tetanus toxoid

1. Introduction the influence of the new conjugation method on the adjuvant activity and toxicity of meningococcal LPS. Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. In particular Neisseria meningitidis LPS plays a role as an immunogen 2. Materials and methods and adjuvant in vaccines based on outer membrane vesicles. When it is free, its toxicity results in significant and unac- Meningococcal LPS and TT were obtained from manu- ceptable reactogenicity. Studies have been directed to reduce facturing facilities of the Finlay Institute, Cuba. LPS (5 mg) LPS endotoxicity and to retain its adjuvant activity [1,2]. was conjugated to TT (25 mg) by the new method using A new method to conjugate polysaccharides, developed in 1-ethyl-3(3-dimethylaminopropyl) carbodiimide [3]. In vitro our department involves the activation of polysaccharides endotoxin activity was tested by the Limulus amoebocyte by basic hydrolysis with the generation of amine groups (LAL) test (Biowittaker, USA). In vivo endotoxin was in the saccharidic chain, and the linkage of these groups to evaluated in an endotoxic shock model. BALB/c mice carboxyl groups of carriers like tetanus toxoid (TT) by a (CENPALAB, Cuba), 6 weeks old were inoculated by the carbodiimide-mediated reaction. In this study, we evaluated intraperitoneal route with one dose of 1 mg of native LPS or activated LPS. Survival was determined after five days. BALB/c mice (CENPALAB, Cuba), 6 weeks old (n = 5 per ∗ Corresponding author. group) were immunized subcutaneously on days 0 and 28 E-mail address: jbalboa@finlay.edu.cu (J.A. Balboa). with treated LPS (2 ␮g), TT (6 ␮g) or conjugated LPS–TT

Table 1 Serum anti lipopolysaccharide (LPS) or tetanus toxoid (TT) antibody subclasses after immunization of mice with meningococcal LPS-TT conjugate, TT or detoxiﬁed LPS (LPSd) Immunogen Day Absorbance (492 nm) at 1:100 dilution

TT LPSn

IgG1 IgG2a IgG1 IgG2a Men LPS-TT 0 0.15 (0.034)a 0.23 (0.028) 0.15 (0.034) 0.13 (0.028) 35 1.61 (0.221) 1.70 (0.254) 1.60 (0.251) 1.51 (0.214) TT 0 0.22 (0.013) 0.25 (0.015) – – 35 1.73 (0.201) 1.16 (0.135) – – LPSd 0 – – 0.09 (0.013) 0.09 (0.015) 35 – – 0.10 (0.051) 0.18 (0.035) a Results are expressed as the optical density mean for groups of ﬁve animals and the S.E. are shown in parentheses.

(2 ␮g), Mice were bled on day 35 and sera were collected and LPS-deﬁcient outer membrane complexes [5]. In conclusion, stored at −20 ◦C. Antibody levels against TT or LPS were the conjugation method reduces the toxicity of LPS, increases assayed by enzyme-linked immunosorbent assay (ELISA) its immunogenicity and retains its strong adjuvant activity. using polystyrene microplates (Costar, USA), TT or LPS at 20 ␮g/mL and sera samples at 1:100 dilution. The procedure was as described previously [4]. References

[1] Petrov AB, Semenov BF, Vartanyan YP, Zakirov MM, Torchilin VP, 3. Results and discussion Trubetskoy VS, et al. Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes. Infect The endotoxic activity of treated LPS was reduced 2400 Immun 1992;60(9):3897–903. ␮ [2] Van der Ley P, Steeghs L, Hamstra HJ, ten Hove J, Zomer B, van times as determined by LAL assay (12,140–5 EU/ g) and Alphen L. Modification of lipid A biosynthesis in Neisseria meningi- no mortality was observed in Balb/c mice inoculated with tidis lpxL mutants: influence on lipopolysaccharide structure, toxicity, detoxified LPS versus 100% mortality in native LPS inoc- and adjuvant activity. Infect Immun 2001;9(10):5981–90. ulated mice. A higher anti-LPS IgG response (OD 0.83) [3] Cabrera O, Cuello M, Sierra G, Soto CR, Mart´ınez ME. Metodo´ de in the Meningococcal LPS-TT conjugate immunized mice obtencion´ de vacunas conjugadas. Composiciones vacunales. Patent apply PCT/CU 03/00013. than unconjugated LPS immunized mice (OD 0.30) was in- [4] Ferriol X, Garc´ıa AG, Ochoa R, Bravo I, Blanco R, Estrada E. Val- duced. Both conjugated and free TT immunized groups had idacion´ de un ELISA para la cuantificacion´ de IgG humana anti high levels of anti-TT IgG (OD 1.31 and OD 1.70, respec- prote´ına de Neisseria meningitidis serogrupo B. Rev Cubana Med tively). However, the level of anti-TT IgG2a was higher in Trop 1999;51(2):99–105. animals immunized with the conjugate than unconjugated TT [5] Steeghs L, Kuipers B, Hamstra HJ, Kersten G, van Alphen L, van der Ley P. Immunogenicity of outer membrane proteins in (Table 1). This is in contrast to the reduced adjuvant activity a lipopolysaccharide-deficient mutant of Neisseria meningitidis: in- of alkali-hydrolyzed meningococcal LPS observed in exper- fluence of adjuvants on the immune response. Infect Immun iments to test its potential to enhance the immunogenicity of 1999;67(10):4988–93.